The advent of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) techniques, coupled with improved performance and lower cost of mass analyzers, has in the past decade allowed mass spectrometry (MS) to take a prominent place among analytical tools used in the study of biologically relevant macromolecules, such as proteins.
For example, in a technique known as peptide mass fingerprinting, mass spectrometry is used to identify proteins purified from biological samples. Identification is effected by matching the mass spectrum of proteolytic fragments of the purified protein with masses predicted from primary sequences prior-accessioned into a database. Roepstorff, The Analyst 117:299-303 (1992); Pappin et al., Curr. Biol. 3(6):327-332 (1993); Mann et al., Biol. Mass Spectrom. 22:338-345 (1993); Yates et al., Anal. Biochem. 213:397-408 (1993); Henzel et al., Proc. Natl. Acad. Sci. USA 90:5011-5015 (1993); James et al., Biochem. Biophys. Res. Commun. 195:58-64 (1993).
Mass spectrometric techniques have also been developed that permit at least partial de novo sequencing of isolated proteins. Chait et al., Science 262:89-92 (1993); Keough et al., Proc. Natl. Acad. Sci. USA. 96:7131-6 (1999); reviewed in Bergman, EXS 88:133-44 (2000).
Additional analytical power has been achieved through introduction of MS/MS analysis, using fragment mass spectra obtained from either MALDI post-source decay (PSD) or collision induced dissociation (CID). Eng et al., J. Am. Soc. Mass. Spectrom. 5:976-989 (1994); Griffin et al., Rapid Commun. Mass Spectrom. 9:1546-1551 (1995); Yates et al., U.S. Pat. Nos. 5,538,897 and 6,017,693; Mann et al., Anal. Chem. 66:4390-4399 (1994).
MALDI time-of-flight (TOF) PSD analysis relies on metastable decay in the drift region to produce daughter ions from selected parents; as a result, the degree of fragmentation is difficult to control or predict. It also suffers from poor sensitivity and mass accuracy. Recently, Loboda et al., Rapid Communic. Mass Spectrom. 14:1047-1057 (2000), described a tandem quadrupole TOF mass spectrometer having a MALDI source, which brings the advantages of CID MS/MS analysis to MALDI-sourced samples.
Further improvement in the mass spectrometric study of complex inhomogeneous biological samples has come from the development of affinity capture laser desorption ionization (LDI) approaches. Hutchens et al., Rapid Commun. Mass Spectrom. 7: 576-580 (1993); U.S. Pat. Nos. 5,719,060, 5,894,063, 6,020,208, 6,027,942, and 6,225,047.
In affinity capture LDI, laser desorption probes are used that have an affinity reagent on at least one surface. The affinity reagent adsorbs desired analytes from heterogeneous samples, concentrating them on the probe surface in a form suitable for subsequent laser desorption ionization. The direct coupling of adsorption and desorption of analyte obviates off-line purification approaches, permitting analysis of smaller initial samples and further facilitating sample modification approaches directly on the probe surface prior to mass spectrometric analysis.
Merchant et al., Electrophoresis 21:1164-1167 (2000), describe a tandem quadrupole/time-of-flight mass spectrometer adapted to use affinity capture probes, coupling high mass accuracy CID MS/MS analysis to affinity capture laser desorption ionization probe techniques.
The MALDI and affinity capture LDI tandem mass spectrometers described, respectively, in Loboda et al. and Merchant et al., extract ions orthogonally into the time of flight detector. Orthogonal extraction serves to decouple the desorption process from the mass analysis, which makes calibration simpler and more stable, sample handling more flexible, and provides other advantages over parallel (axial) injection, even in single MS mode.
However, in contrast to parallel ion extraction geometries, for which ions need survive only on the order of 10-300 microseconds before TOF analysis and detection, orthogonal acceleration TOF requires the formation of ions that must survive for at least 2-3 msec prior to TOF analysis and ultimate detection. See Krutchinksy et al., Rapid Commun. Mass Spectrometry, 12: 508-518 (1998); Chernushevich et al., “Orthogonal-Injection TOFMS for Analyzing Biomolecules”, Anal. Chem. 71, 452A-461A (Jul. 1, 1991).
For various biopolymers such as proteins and peptides, ion stability, and thus ultimate survival time, depends on a number of factors, including:
(1) nascent bond energies of amino acid residues and other constituents;
(2) initial thermal energies of desorption;
(3) initial thermal energies of ionization;
(4) energies and frequency of post desorption collisions; and
(5) gas phase reactions following desorption/ionization.
The nascent bond energies being inherent in the biomolecule to be analyzed, there is thus a need in the art for apparatus and methods that increase ion survival times by reducing the initial thermal energies of desorption, reducing the initial thermal energies of ionization, decreasing the energies and frequencies of post desorption collisions, and/or by reducing gas phase degradative reactions.